1. Field of the Invention
This invention relates to methods for administering biologically active peptides to a mammalian host by the introduction thereto of one or more polynucleotides to operatively encode for the peptides, preferably by non-invasive means. It also relates to the administration of said polynucleotides to prevent and treat illnesses and loss of immune function associated with aging in mammals.
2. Description of Related Art
The direct introduction of a biologically active peptide or protein into the cells of a patient can have significant therapeutic value. However, this approach also has several drawbacks. Of primary concern is the risk of potential toxicities, particularly at dosages sufficient to produce a biological response to the peptide. From a practical perspective, there is also the problem of the cost associated with isolating and purifying or synthesizing the peptides. Moreover, the clinical impact of the peptides is also limited by their relatively short half-life in vivo which usually results from their degradation by any proteases present in the target tissue.
For these reasons, introduction of a protein into a patient by delivery of a gene which will express the protein in the patient/host is an intriguing alternative to administering the protein. However, to date the principal means for introduction of foreign genetic material into a host has involved the integration of the gene into the host genome by, for example, transforming the host's cells with a viral vector. Direct in vivo gene transfer into postnatal animals has also been reported using DNA encapsulated in liposomes including DNA entrapped in proteoliposomes containing viral envelope receptor proteins.
In 1984, work at the NIH was reported which showed that intrahepatic injection of naked, cloned plasmid DNA for squirrel hepatitis into squirrels produced both viral infection and the formation of antiviral antibodies in the squirrels (Seeger, et al., Proc.Nat'l.Acad.Sci USA, 81:5849-5852, 1984). Several years later, Feigner, et al., reported that they obtained expression of protein from "naked" polynucleotides (i.e., DNA or RNA not associated with liposomes or a viral expression vector) injected into skeletal muscle tissue (Felgner, et al., Science, 247:1465, 1990; see also, PCT application WO 90/11092). Felgner, et al. surmised that muscle cells efficiently take up and express polynucleotides because of the unique structure of muscle tissue, which is comprised of multinucleated cells, sarcoplasmic reticulum and a transverse tubular system which extends deep into the muscle cell.
Although it has been supposed that cells of other tissues may also be able to take up naked polynucleotides, expression in other tissues has only been identified to date when delivery of the expressed gene was via a delivery system, e.g., liposomal transformation of the cells. Indeed, other researchers have suggested that uptake and expression of naked polynucleotides in tissues other than skeletal musde does not occur at detectable or biologically active levels (see, e.g., Stribling, et al., Proc. Natl. Acad. Sci. USA, 89:11277-11281, 1992 [expression following aerosol delivery of a gene occurred with use of a liposomal delivery system but not with introduction of DNA alone]; and, Tang, et al., Nature, 356:152-154, 1992 [injection with a vaccine "gun" of an hGH plasmid coupled to colloidal gold beads into the skin of mice did not elicit an immune response]).
Although generally effective for gene expression within muscle cells, injection of DNA or RNA into muscle tissue for long-term therapy requires use of repeated injections to offset loss of expression from gene degradation. This approach may not only be time-consuming and expensive, but may also be impractical due to inflammation caused at and near the site of injection. Such inflammation can cause muscle or other somatic cells into which nucleotides are introduced to be themselves targeted by an immune response (see, e.g., Example I) and can lead to severe myonecrosis. Further, intramuscular injection of DNA not only risks injury to muscle tissue, but that injury apparently also compromises the efficacy of the therapy. For example, researchers working with the University of Ottawa recently observed that "[s]triated muscle is the only tissue found to be capable of taking up and expressing reporter genes that are transferred in the form of plasmid DNA . . . but our findings indicate that fibers damaged by the injection procedure do not take up and express plasmid DNA." (Davis, et al., Human Gene Therapy, 4:151-159, 1993).
Further, while use of intramuscular injections may be effective on at least a short term basis in therapies directed to disease in the muscle tissue itself, it is likely to be less effective in stimulating a tissue specific immune or other biological response to the expressed peptide elsewhere in the patient's body.
As a result, intramuscular injection is not a particularly viable route for achieving expression of peptides at the primary entry points for many infections; i.e., skin and mucosa.
Further, it appears that intramuscular injections of polynucleotides will lead to the formation of both antibodies and cytotoxic T cells in the tissue, due to release of any encoded protein by targeted muscle cells. In contrast, injection of protein (e.g., in a vaccination scheme) does not usually induce cytotoxic T cell formation because exogenous proteins do not efficiently enter the class I processing pathway.
In PCT application WO 90/11092 (discussed supra), the inventors propose tbat the injection of naked DNA into skeletal muscle or other somatic issues will lead to direct gene expression in the cytoplasm of the injected cells. The inventors further suppose that the encoded protien will then enter the class I processing pathway to induce cytotoxic T cell formation (which are necessary for the control of established viral infections and cancers). However, as discussed above, it appears that instead any somatic cell that expresses antigen must first release the antigen into the extracellular space for uptake by antigen presenting cells before a class I restricted cytotoxic T cell response can to the antigen can be induced. This conclusion is supported by recent research regarding antigen presentation where the observation was made that "the priming of an immune response against . . . class I restricted antigen that is expressed exclusively in non-hematopoietic cells invovlves the transfer of that antigen to a host bone marrow derived cell before its presentation." (Huang, et al., Science, 264:961-965, 1994). Thus, at least one premise on which the method for introduction of genetic material into muscle cells for protein expression of PCT application WO 90/11092 was based may not be accurate.
Use of intramuscular injections can, however, produce relatively high levels of protein expression systemically prior to degradation of the injected gene. While this response is desirable in therapies where protein replacement is the goal, it can lead to unintended toxicities in immunization protocols where relatively rapid clearance or lower levels of expression are optimal. As a result, introduction of the gene into tissues which regularly shed or regenerate (such as skin) and/or into cells with a relatively high attrition rate in vivo (such as antigen presenting cells) would be more useful routes for gene immunization.
With respect to delivery systems for genes, means such as viral vectors which introduce the gene into the host's genome present potential health risks association with damage to the genetic material in the host cell. Use of cationic liposomes or a biolistic device (i.e., a vaccine "gun" which "shoots" polynucleotides coupled to beads into tissue) to deliver genes in vivo is preparation intensive and requires some experimentation to select proper particle sizes for transmission into target cells. Further, any invasive means of introducing nucleotides (e.g., injection) poses problems of tissue trauma (particularly in long-term therapies) and presents limited access to certain target tissues, such as organs.
Means for non-invasive delivery of pharmaceutical preparations of peptides, such as iontophoresis and other means for transdermal transmission, have at least the advantage of minimizing tissue trauma. However, it is believed that the bioavailability of peptides following transdermal or mucosal transmission is limited by the relatively high concentration of proteases in these tissues. Yet unfortunately, reliable means of delivering peptides by transdermal or mucosal transmission of genes encoding for them has been unavailable.
The potential benefits of successful administration of peptides via in vivo expression of naked genes can be illustrated by comparison to the present state of immunotherapy wherein cytokine proteins (such as interleukin-2, hereafter "IL-2") are administered to a patient to treat or prevent diseases associated with aging.
Certain diseases occur as part of the aging process in virtually all mammalian species, despite differing life styles and life spans among those species. These diseases include cancer, hypertension, vascular diseases and insulin resistance that can result in diabetes. Because the timing of the onset of these diseases cannot be solely attributed to environmental factors, it has been assumed that their onset is genetically programmed. However, the processes which actually control the aging process and the incidence of age-associated illnesses are not known.
In general, aging is associated with a reduced ability to mount an immune response to exogenous antigens, a decreased functional reserve and response to stress, as well as an increased tendency toward fibrosis. All of these states are contributed to or controlled in part in vivo by circulating cytokines.
For example, interleukin-1 (IL-1) proteins affect glucose homeostasis and can act as a hypoglycemic agent in insulin resistant C57BL/Ks db mice and C57BL/6G ob/ob mice (Del Rey, et al., Proc. Natl. Acad. Sci., USA, 86:5943, 1989). In these animal models of adult onset diabetes, a single injection of human recombinant IL-1 normalized glucose blood levels for several hours. IL-1 is also known to exert effects on the hypothalamic-pituitary axis that influences appetite, and the response to stress. Thus, abnormalities in IL-1 production or responsiveness could underlie both non-insulin dependent diabetes and obesity in aging. IL-1 gene therapy (administered exactly as described for IL-2) may, therefore, prevent the onset of diabetes in mice and humans.
High blood pressure is another concomitant of aging that is frequently associated with diabetes. Recent experiments have shown that a major regulator of blood pressure is the endothelium-derived relaxing factor, nitric oxide. The production of nitric oxide is regulated by IL-1. Hence, the increase in blood pressure that occurs with aging may also be prevented by IL-1 gene therapy. IL-1 protein induces fever and even shock when administered acutely to animals. However, the continuous production of low levels of IL-1 following somatic gene therapy will avoid these side effects.
The biological effects of administering pharmaceutical doses of cytokine proteins have been explored recently by researchers seeking to stimulate the immune system to augment its response to certain pathogens and to maintain immune function in immunodeficient patients, such as those infected with human immunodeficiency virus (HIV).
Specifically, efforts to administer IL-2 as a therapeutic in immune system diseases have been recently reported by Teppler, et al., J. Exp. Med., 177:483-492, 1993, (administration of recombinant IL-2 protein conjugates of polyethylene glycol to HIV infected patients); Caligiuri, et al., J. Clin. Invest., 91:123-132, 1993, (prolonged infusions of recombinant IL-2 protein to patients with advanced cancers), and Kaplan, et al., i Bio/Technology 10:157-162, 1992, (administration of IL-2 to patients infected with M. leprae or HIV). The focus of these studies has been to develop means of administering the IL-2 protein in a way which will minimize its toxicity.
Toxicity of the IL-2 protein has been a major impediment to its use as an effective therapeutic. To be effective, IL-2 therapy generally requires that the administered protein be present in serum in sufficient quantity to saturate high affinity IL-2 receptors and induce marked expansion of circulating natural killer (NK) cells. With high doses of IL-2, however, come life-threatening toxicities such as severe hypotension, pulmonary edema, renal failure, cardiac arrhythmias and neurologic disfunction.
The approach taken by the above-referenced researchers to overcome this problem has been to experiment with prolonged administration of IL-2 protein at relatively low doses after the onset of infection and/or disease. This approach, however, has yet to define effective parameters for consistent, predictable IL-2 therapy. In particular, the levels of circulating protein resulting from introduction of cytokines vary substantially over time, in part due to protease degradation. Repeated injections are, therefore, required with resulting "hills and valleys" in the quantity of protein available to the patient. Moreover, the work with IL-2 does not indicate whether a similar approach would, even after substantial clinical experimentation, prove effective for use with other cytokines which play roles in various disease states, including age-associated illnesses.
A need, therefore, exists for an efficient means of introducing a protein (including but not limited to cytokines and antigens) into a host in a manner which will minimize the toxicity of the protein. More specifically, a need also exists for a means for introducing a protein into a host in a manner which will produce a consistent but subtherapeutic level of protein expression over a long period of time. In the latter respect, a need particularly exists for a means of administering cytokines to prevent as well as treat age-associated illnesses.
More generally, the above discussion also illustrates the need for an effective means of introducing naked nucleotides which will express in vivo a peptide which can induce local immunity in skin and mucosa to vaccinate a host against, for example, sexually transmitted diseases and respiratory illnesses.
It also suggests a need for a means of introducing a gene encoding for a biologically active peptide to a host in a tissue-specific manner without significant tissue trauma.
The present invention addresses all of these needs.